Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
  • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
  • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/104—Pseudomonadales, e.g. Pseudomonas
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/02—Acyclic radicals, not substituted by cyclic structures
  • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
  • C07K16/1214—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pseudomonadaceae (F)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2469/00—Immunoassays for the detection of microorganisms
  • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

• the present invention relates to immunogenic and immunoprotective compositions and methods for making and using homogenous synthetic P. aeruginosa lipooligosaccharide (LOS)-based oligosaccharides, conjugates, and antibodies derived therefrom.
• LOS homogenous synthetic P. aeruginosa lipooligosaccharide
• the present invention provides synthetic oligosaccharide 1a or 1 b:
• R 1 and R 2 are each independently H, ⁇ -Rha-, ⁇ -Glc(1-2)- ⁇ -Rha-, ⁇ -QuiNAc(1-3)- ⁇ -Rha-, ⁇ -FucNAc(1-3)- ⁇ -Rha-, ⁇ -Rha[2,3,4-OAc]-, ⁇ -QuiNAc(1-3)- ⁇ -Rha[2,4-OAc]-, or ⁇ -FucNAc(1-3)- ⁇ -Rha[2,4, -OAc]-.
• R 1 and R 2 are selected from the combinations in the following table:
• R 1 R 2 ⁇ -Rha- H ⁇ -Glc(1-2)- ⁇ -Rha- H H ⁇ -Rha- H ⁇ -QuiNAc(1-3)- ⁇ -Rha- H ⁇ -FucNAc(1-3)- ⁇ -Rha- H ⁇ -Rha[2,3,4-OAc]- H ⁇ -QuiNAc(1-3)- ⁇ -Rha[2,4-OAc]- H ⁇ -FucNAc(1-3)- ⁇ -Rha[2,4,-OAc]-
• the present invention also includes compositions comprising an antigen 1a and/or 1 band a pharmaceutically acceptable vehicle.
• the composition contains a single antigen or a known, defined mixture of antigens.
• the invention further provides vaccine compositions, including immunogenic and immunoprotective compositions, comprising antigen 1a and/or 1 b and a pharmaceutically acceptable vehicle.
• These vaccine compositions can optionally include a pharmaceutically acceptable adjuvant.
• the vaccine compositions are endotoxin-free.
• the vaccine compositions can be mono-, di-, tri- or tetravalent.
• the invention further provides a method for synthetically forming oligosaccharides 1a and antigens 1b.
• the invention further provides methods for diagnosing, treating, and preventing infections caused by P. aeruginosa.
• oligosaccharide refers to a compound containing two or more monosaccharide units. Oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not the monosaccharide unit at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, oligosaccharides are depicted herein with the non-reducing end on the left and the reducing end on the right.
• oligosaccharides described herein are described with the name or abbreviation for the non-reducing monosaccharide (e.g., Gal), preceded by the configuration of the glycosidic bond ( ⁇ or ⁇ ), the ring bond, the ring position of the reducing monosaccharide involved in the bond, and then the name or abbreviation of the reducing monosaccharide (e.g., GlcNAc).
• the linkage between two sugars may be expressed, for example, as 2,3, 2 ⁇ 3, or 2-3.
• Each monosaccharide is a pyranose or furanose.
• “monosaccharide” or “monosaccharide unit” refers to a single sugar residue in an oligosaccharide, including derivatives therefrom.
• an individual monomer unit is a monosaccharide which is (or can be) bound through a hydroxyl group to another monosaccharide.
• endotoxin-free refers to an oligosaccharide that does not contain endotoxins or endotoxin components normally present in isolated bacterial carbohydrates and polysaccharides.
• synthetic refers to material which is substantially or essentially free from components, such as endotoxins, glycolipids, unrelated oligosaccharides, etc., which normally accompany a compound when it is isolated.
• synthetic compounds are at least about 90% pure, usually at least about 95%, and preferably at least about 99% pure. Purity can be indicated by a number of means well known in the art. Preferably, purity is measured by HPLC. The identity of the synthetic material can be determined by mass spectroscopy and/or NMR spectroscopy.
• linker refers to either a bond or a moiety which at one end exhibits a grouping able to enter into a covalent bonding with a reactive functional group of the carrier, e.g. an amino, thiol, or carboxyl group, and at the other end a grouping likewise able to enter into a covalent bonding with a hydroxyl group or an amino group of an oligosaccharide according to the present invention.
• a biocompatible bridging molecule of suitable length, e.g.
• Linkers preferably include a substituted or unsubstituted (C 1 -C 10 ) alkylene group or a substituted or unsubstituted (C 2 -C 10 ) alkenylene group.
• carrier refers to a protein, peptide, lipid, polymer, dendrimer, virosome, virus-like particle (VLP), or combination thereof, which is coupled to the oligosaccharide to enhance the immunogenicity of the resulting oligosaccharide-carrier conjugate to a greater degree than the oligosaccharide alone.
• VLP virus-like particle
• protein carrier refers to a protein, peptide or fragment thereof, which is coupled or conjugated to an oligosaccharide to enhance the immunogenicity of the resulting oligosaccharide-protein carrier conjugate to a greater degree than the oligosaccharide alone.
• the protein carrier may serve as a T-dependent antigen which can activate and recruit T-cells and thereby augment T-cell dependent antibody production.
• conjugated refers to a chemical linkage, either covalent or non-covalent, that proximally associates an oligosaccharide with a carrier so that the oligosaccharide conjugate has increased immunogenicity relative to an unconjugated oligosaccharide.
• conjugate refers to an oligosaccharide chemically coupled to a carrier through a linker and/or a cross-linking agent.
• passive immunity refers to the administration of antibodies to a subject, whereby the antibodies are produced in a different subject (including subjects of the same and different species) such that the antibodies attach to the surface of the bacteria and cause the bacteria to be phagocytosed or killed.
• protective immunity means that a vaccine or immunization schedule that is administered to a animal induces an immune response that prevents, retards the development of, or reduces the severity of a disease that is caused by a pathogen or diminishes or altogether eliminates the symptoms of the disease.
• Protective immunity may be predicted based on the ability of serum antibody to activate complement-mediated bactericidal activity or confer passive protection against a bacterial infection in a suitable animal challenge model.
• immunoprotective composition refers to a composition formulated to provide protective immunity in a host.
• a sufficient amount to elicit an immune response or “in an effective amount to stimulate an immune response” (e.g., to epitopes present in a preparation) means that there is a detectable difference between an immune response indicator measured before and after administration of a particular antigen preparation.
• Immune response indicators include but are not limited to: antibody titer or specificity, as detected by an assay such as enzyme-linked immunoassay (ELISA), bactericidal assay (e.g., to detect serum bactericidal antibodies), flow cytometry, immunoprecipitation, Ouchter-Lowry immunodiffusion; binding detection assays of, for example, spot, Western blot or antigen arrays; cytotoxicity assays, and the like.
• an assay such as enzyme-linked immunoassay (ELISA), bactericidal assay (e.g., to detect serum bactericidal antibodies), flow cytometry, immunoprecipitation, Ouchter-Lowry immunodiffusion
• binding detection assays of, for example, spot, Western blot or antigen arrays
• cytotoxicity assays and the like.
• antibody encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, F(ab′) 2 fragments, F(ab) molecules, Fv fragments, single chain fragment variable displayed on phage (scFv), single domain antibodies, chimeric antibodies, humanized antibodies, and functional fragments thereof which exhibit immunological binding properties of the parent antibody molecule.
• “monoclonal antibody” refers to an antibody composition having a homogeneous antibody population.
• the term is not limited by the manner in which it is made.
• the term encompasses whole immunoglobulin molecules, as well as Fab molecules, F(ab′) 2 fragments, Fv fragments, single chain fragment variable displayed on phage (scFv), and other molecules that exhibit immunological binding properties of the parent monoclonal antibody molecule.
• the specified antibody or antibodies bind(s) to a particular antigen or antigens in a sample and does not bind in a significant amount to other molecules present in the sample.
• Specific binding to an antibody under such conditions may require an antibody or antiserum that is selected for its specificity for a particular antigen or antigens.
• antigen refers to any substance that may be specifically bound by an antibody molecule.
• immunogen and “immunogenic composition” refer to an antigenic composition capable of initiating lymphocyte activation resulting in an antigen-specific immune response.
• epitope refers to a site on an antigen to which specific B cells and/or T cells respond.
• the term is also used interchangeably with “antigenic determinant” or “antigenic determinant site.”
• B cell epitope sites on proteins, oligosaccharides, or other biopolymers may be composed of moieties from different parts of the macromolecule that have been brought together by folding. Epitopes of this kind are referred to as conformational or discontinuous epitopes, since the site is composed of segments of the polymer that are discontinuous in the linear sequence but are continuous in the folded conformation(s). Epitopes that are composed of single segments of biopolymers or other molecules are termed continuous or linear epitopes.
• T cell epitopes are generally restricted to linear peptides. Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
• Ac means acetyl (—C(O)CH 3 ).
• TBS means tert-butyldimethylsilyl.
• Troc means 2,2,2-trichloroethoxycarbonyl.
• TCI means trichloroacetimidate
• Phth means phthaloyl
• TFA means trifluoroacetate
• TCA means trichioroacetate
• Cbz means benzyloxycarbonyl.
• Bz means benzoyl
• Bn means benzyl
• TES means triethylsilyl
• TBDPS means tert-butyldiphenylsilyl.
• MCA monochioroacetate
• Lev levulinoyl
• ADMB means 4-O-acetyl 2,2dimethylbutanoate.
• Tr triphenylmethyl
• DMT dimethoxytrityl
• FMOC means 9-fluorenylmethyl carbonate.
• Alloc means Allyloxycarbonyl.
• Nap napthyl
• SEt means thioethyl
• SPh means thiophenyl
• STol means thiotolyl
• SAdm means thioadamantyl
• the present invention provides compositions and methods for chemically synthesizing antigenic structures corresponding to a portion of the P. aeruginosa lipooligosaccharide (LOS), a major surface component of the outer membrane.
• LOS P. aeruginosa lipooligosaccharide
• oligosaccharides may include one or more monosaccharide units linked to one another through one or more ⁇ - and/or ⁇ -glycosidic bonds.
• the oligosaccharides will include monosaccharides and glycosidic linkages naturally found in P. aeruginosa LOS structures, generally in 1-2 or 1-4 connectivities.
• the invention further contemplates other connectivities, such as 1-3 and 1-6, especially where the oligosaccharide design is extended beyond naturally P. aeruginosa LOS structures.
• R 1 or R 2 When either of R 1 or R 2 is an oligosaccharide, it may contain between 1 to about 6, preferably up to about 4, monosaccharide units.
• the invention contemplates inclusion of natural and modified monosaccharide units, such as glucose, fucosamine and rhamnose, especially where the oligosaccharide design is extended beyond naturally P. aeruginosa LOS structures.
• preferred saccharide substituents include ⁇ -Rha-(rhamnosyl), ⁇ -Glc(1-2)- ⁇ -Rha-, ⁇ -QuiNAc(1-3)- ⁇ -Rha-, ⁇ -FucNAc(1-3)- ⁇ -Rha-, ⁇ -Rha[2,3,4-OAc]-, ⁇ -QuiNAc(1-3)- ⁇ -Rha[2,4-OAc]-, and ⁇ -FucNAc(1-3)- ⁇ -Rha[2,4, -OAc]-.
• the present invention provides oligosaccharides 1a:
• R 1 and R 2 are independently H, a monosaccharide or an oligosaccharide, and X is H, a linker group or a protecting group.
• R 1 and R 2 are selected from H, ⁇ -Rha- (rhamnosyl), ⁇ -Glc(1-2)- ⁇ -Rha-, ⁇ -QuiNAc(1-3)- ⁇ -Rha-, ⁇ -FucNAc(1-3)- ⁇ -Rha-, ⁇ -Rha[2,3,4-OAc]-, ⁇ -QuiNAc(1-3)- ⁇ -Rha[2,4-OAc]-, and ⁇ -FucNAc(1-3)- ⁇ -Rha[2,4, -OAc]-. More preferably, R 1 and R 2 are selected from the combinations in the table below:
• R 1 R 2 ⁇ -Rha- H ⁇ -Glc(1-2)- ⁇ -Rha- H H ⁇ -Rha- H ⁇ -QuiNAc(1-3)- ⁇ -Rha- H ⁇ -FucNAc(1-3)- ⁇ -Rha- H ⁇ -Rha[2,3,4-OAc]- H ⁇ -QuiNAc(1-3)- ⁇ -Rha[2,4-OAc]- H ⁇ -FucNAc(1-3)- ⁇ -Rha[2,4,-OAc]-
• the present invention provides oligosaccharides 1b:
• R 1 and R 2 are independently H, a monosaccharide or an oligosaccharide, L is a linker group, and Y is a carrier.
• R 1 and R 2 are selected from H, ⁇ -Rha- (rhamnosyl), ⁇ -Glc(1-2)- ⁇ -Rha-, ⁇ -QuiNAc(1-3)- ⁇ -Rha-, ⁇ -FucNAc(1-3)- ⁇ -Rha-, ⁇ -Rha[2,3,4-OAc]-, ⁇ -QuiNAc(1-3)- ⁇ -Rha[2,4-OAc]-, and ⁇ -FucNAc(1-3)- ⁇ -Rha[2,4, -OAc]-. More preferably, R 1 and R 2 are selected from the combinations in the table below:
• R 1 R 2 ⁇ -Rha- H ⁇ -Glc(1-2)- ⁇ -Rha- H H ⁇ -Rha- H ⁇ -QuiNAc(1-3)- ⁇ -Rha- H ⁇ -FucNAc(1-3)- ⁇ -Rha- H ⁇ -Rha[2,3,4-OAc]- H ⁇ -QuiNAc(1-3)- ⁇ -Rha[2,4-OAc]- H ⁇ -FucNAc(1-3)- ⁇ -Rha[2,4,-OAc]-
• oligosaccharide (1a) has formula (2a):
• oligosaccharide (1 b) has formula (2b):
• oligosaccharide (1a) has formula (3a):
• oligosaccharide (1b) has formula (3b):
• oligosaccharide (1a) has formula (4a):
• oligosaccharide (1b) has formula (4b):
• oligosaccharide (1a) has formula (5a):
• oligosaccharide (1 b) has formula (5b):
• oligosaccharide (1a) has formula (6a):
• oligosaccharide (1b) has formula (6b):
• oligosaccharide (1a) has formula (7a):
• oligosaccharide (1b) has formula (7b):
• oligosaccharide (1a) has formula (8a):
• oligosaccharide (1 b) has formula (8b):
• oligosaccharide (1a) has formula (9a):
• oligosaccharide (1b) has formula (9b):
• Table 1 lists preferred R 1 and R 2 combinations and Psuedomonas serotypes proposed to be covered by the permutations.
• the present invention provides LOS antigens comprised of core oligosaccharide structures or motifs corresponding to one or more of the 20 major serotypes, members within a given serotype, and individual serotype subtypes as depicted in Table 1.
• the linker is exemplified as an alkylenethiol group having between 1 and 20 carbon atoms, preferably between 1 and 8.
• the linker shown i.e., the alkylenethiol group
• the present invention contemplates and provides sufficient guidance below for modifying any of the above-described thiol products with different linkers and/or spacers, and to make LOS structures directed to any of the oligosaccharides described above, including any subsequence combinations derived therefrom, or indeed, any P. aeruginosa LOS structure for that matter.
• the invention provides polyvalent LOS antigen combinations (and conjugates thereof) representing pluralities of any of the different oligosaccharides described in Table 1, for example, or multivalent combinations of one or more of the synthetic oligosaccharides 1a or 1b with one or more other protein antigens, carbohydrate 0-antigens, and/or alginates
• Suitable linkers comprise at one end a grouping able to enter into a covalent bonding with a reactive functional group of the carrier, e.g. an amino, thiol, or carboxyl group, and at the other end a grouping likewise able to enter into a covalent bonding with a hydroxyl group of an oligosaccharide according to the present invention.
• a reactive functional group of the carrier e.g. an amino, thiol, or carboxyl group
• a grouping likewise able to enter into a covalent bonding with a hydroxyl group of an oligosaccharide according to the present invention.
• a biocompatible bridging molecule of suitable length e.g. substituted or unsubstituted heteroalkylene, arylalkylene, alkylene, alkenylene, or (oligo)alkylene glycol groups.
• Linkers preferably include substituted or unsubstituted alkylene or alkenylene groups containing 1
• Linkers able to react with thiol groups on the carrier are, for example, maleimide and carboxyl groups; preferred groupings able to react with aldehyde or carboxyl groups are, for example, amino or thiol groups.
• Preferred covalent attachments between linkers and carriers include thioethers from reaction of a thiol with an ⁇ -halo carbonyl or ⁇ -halo nitrile, including reactions of thiols with maleimide; hydrazides from reaction of a hydrazide or hydrazine with an activated carbonyl group (e.g. activated NHS-ester or acid halide); triazoles from reaction of an azide with an alkyne (e.g.
• linker molecules are known to skilled workers and commercially available or can be designed as required and depending on the functional groups present and can be prepared by known methods.
• Suitable carriers are known in the art (See e.g., Remington's Pharmaceutical Sciences (18th ed., Mack Easton, Pa. (1990)) and may include, for example, proteins, peptides, lipids, polymers, dendrimers, virosomes, virus-like particles (VLPs), or combinations thereof, which by themselves may not display particular antigenic properties, but can support immunogenic reaction of a host to the oligosaccharides of the present invention (antigens) displayed at the surface of the carrier(s).
• VLPs virus-like particles
• the carrier is a protein carrier, including but not limited to, bacterial toxoids, toxins, exotoxins, and nontoxic derivatives thereof, such as tetanus toxoid, tetanus toxin Fragment C, diphtheria toxoid, CRM (a nontoxic diphtheria toxin mutant) such as CRM 197, cholera toxoid, Staphylococcus aureus exotoxins or toxoids, Escherichia coli heat labile enterotoxin, Pseudomonas aeruginosa exotoxin A, including recombinantly produced, genetically detoxified variants thereof; bacterial outer membrane proteins, such as Neisseria meningitidisserotype B outer membrane protein complex (OMPC), outer membrane class 3 porin (rPorB) and other porins; keyhole limpet hemocyanine (KLH), hepatitis B virus core protein, thyroglobulin,
• a carrier may display on average, for example, 1 to 500, 1 to 100, 1 to 20, or 3 to 9 oligosaccharide units on its surface.
• the carbohydrate antigens of the present invention are conjugated to CRM197, a commercially available protein carrier used in a number of FDA approved vaccines.
• CRM-conjugates have the advantage of being easier to synthesize, purify and characterize than other FDA approved carriers such as OMPC.
• Carbohydrate antigens may be conjugated to CRM via thiol-bromoacetyl conjugation chemistry. CRM activation may be achieved by reacting the lysine side chains with the NHS ester of bromoacetic acid using standard conditions as previously described in U.S. Pat. Appl. Publ. 2007-0134762, the disclosures of which are incorporated by reference herein.
• CRM conjugates may be purified via size exclusion chromatography to remove and recover any unreacted carbohydrate.
• MBTH specific for GlcNAc residues
• Bradford assays may be used to determine carbohydrate:protein ratio and protein content, respectively, as previously described (Manzi et al., Curr. Prot. Mol. Biol., section 17.9.1 (Suppl. 32), 1995.
• a minimum carbohydrate content of about 10% by weight for each conjugate may be generated.
• a conjugate may include about 3-20 antigens per protein carrier.
• carbohydrate antigens may be conjugated to one or more carriers suitable for development of diagnostic assays, including ELISAs and microarrays.
• exemplary carriers for use in such assays include bovine serum albumin (BSA), keyhole limpet hemocyanine (KLH), biotin, a label, a glass slide or a gold surface.
• BSA bovine serum albumin
• KLH keyhole limpet hemocyanine
• biotin a label
• glass slide or a gold surface a gold surface.
• synthetic carbohydrate antigens may be conjugated to BSA by a thiol-maleimide coupling procedure (FIG. 5B).
• BSA conjugates may be purified via size exclusion chromatography to remove and recover any unreacted carbohydrate. Characterization via the phenol-sulfuric acid and Bradford assays may be performed along with MALDI-MS to provide information on the carbohydrate content and valency of the conjugates. In preferred embodiments, conjugates will contain aminimum carbohydrate content of about 10% by weight per BSA conjugate and >8 antigen copies per conjugate.
• the present invention provides a method for assembling synthetic homogenous LOS-oligosaccharide structures from P. aeruginosa , including those described above from monosaccharide and disaccharide building blocks.
• the outer core may be prepared according to procedures described in WO2012/082635 following the retrosynthesis in Scheme I.
• ⁇ -Glucosyl donors may be prepared using the following scheme:
• Galactosamine linker units suitable for preparing P. aeruginosa vaccine outer core synthesis may be prepared as follows:
• the hexyl(benzyl)carbamate compound may be prepared using procedures described by Whitfield D. M. et al (Collet. Czech. Chem. Commun. 58:159-17291993)).
• the 6-O and 4-O positions may be protected with benzylidine following Bedini E. et al. Carbohydrate Res. 349:24-32 (2012).
• Branched glucose disaccharide units suitable for preparing P. aeruginosa vaccine outer core may be prepared as follows:
• Core tetrasaccharide assemplies suitable for preparing P. aeruginosa vaccine outer core may be prepared as following:
• the selective coupling of GalN 3 -3,4-diol with the disaccharide may be achieved by following procedures disclosed by Osswald et al. ( Z. Naturforsch. 58b:764-774 (2003)) and Komarova B. S. et al. ( Tetrahed. Lett. 47:3583-78 (2006)). Both references show the selectivity of the reaction in favor of 3-position over the 4-position. Komarova also describes a process for coupling the 4-position of the resulting trimer with OTFI-activated monosaccharide to provide a tetramer.
• compositions and methods for synthesis of the above described LOS-oligosaccharides and conjugates thereof, including others described in Table 1 are described in Examples 1 to 9 below.
• Protecting groups employed in the synthesis of LOS-oligosaccharides may include those customarily considered in sugar chemistry, for example those mentioned in “Protective Groups in Organic Synthesis”, 3 rd edition, T. W. Greene and P. G. M. Wuts (Ed.), John Wiley and Sons, New York, 1999.
• the present invention provides immunogenic and immunoprotective compositions containing LOS oligosaccharides or LOS oligosaccharide-protein carrier conjugates for inducing an immune response to LOS antigens.
• the immunogenic compositions may include one or more adjuvants, as well as pharmaceutically acceptable vehicles suitable for administration to an animal or individual.
• An immunogenic or immunoprotective composition will include a “sufficient amount” or “an immunologically effective amount” of a oligosaccharide-protein carrier conjugate according to the present invention, as well as any of the above mentioned components, for purposes of generating an immune response or providing protective immunity, as further defined herein.
• the invention provides an immunogenic composition comprising one or more LOS oligosaccharide(s) I a or LOS oligosaccharide-protein carrier conjugate(s) 1b suitable for inducing an immune response against P. aeruginosa.
• the invention provides a pharmaceutical composition comprising a LOS oligosaccharide(s) 1a or LOS oligosaccharide-protein carrier conjugate 1 b formulated as a vaccine for protection against P. aeruginosa infections.
• the invention provides a pharmaceutical composition comprising an oligosaccharide-protein carrier conjugate 1 b formulated as a vaccine for protecting against one or more P. aeruginosa serotypes as described herein.
• the invention provides a pharmaceutical composition comprising an antibody and a physiologically acceptable vehicle for use in a method for providing passive immunity or treatment against one or more P. aeruginosa serotypes. More particularly, the invention provides an antibody preparation against one or more LOS-oligosaccharide conjugate 1 b compositions in accordance with the present invention.
• the antibody preparation may include any member from the group consisting of polyclonal antibody, monoclonal antibody, mouse monoclonal IgG antibody, humanized antibody, chimeric antibody, fragment thereof, or combination thereof.
• the invention further contemplates a hybridoma cell producing a monoclonal antibody directed against any of the LOS-oligosaccharide described herein.
• Administration of oligosaccharides or oligosaccharide-protein carrier conjugates or antibodies thereto may be carried out by any suitable means, including by parenteral administration (e.g., intravenously, subcutaneously, intradermally, or intramuscularly); by topical administration, of for example, antibodies to an airway surface; by oral administration; by in ovo injection in birds, for example, and the like.
• parenteral administration e.g., intravenously, subcutaneously, intradermally, or intramuscularly
• topical administration e.g., of for example, antibodies to an airway surface
• oral administration e.g., by in ovo injection in birds, for example, and the like.
• each immunogenic or immunoprotective composition includes one or more oligosaccharide(s) according to Formula 1a or 1 b or conjugates thereof in a pharmaceutically acceptable vehicle or diluent forming a substantially aqueous mixture.
• the immunogenic or immunoprotective compositions includes one or more oligosaccharide-protein carrier conjugates(s) in conjunction with one or more pharmaceutically acceptable adjuvant(s), vehicles and/or protein carriers suitable for administration to an animal or individual.
• An oligosaccharide-protein carrier conjugate composition may further include one or more immunologic adjuvant(s).
• An immunologic adjuvant is a compound that, when combined with an antigen, increases the immune response to the antigen as compared to the response induced by the antigen alone so that less antigen can be used to achieve a similar response.
• an adjuvant may augment humoral immune responses, cell-mediated immune responses, or both.
• adjuvant can overlap to a significant extent.
• a substance which acts as an “adjuvant” may also be a “carrier,” and certain other substances normally thought of as “carriers,” for example, may also function as an “adjuvant.”
• a substance which may increase the immunogenicity of the synthetic oligosaccharide or carrier associated therewith is a potential adjuvant.
• a carrier is generally used in the context of a more directed site-specific conjugation to an oligosaccharide of the present invention, whereby an adjuvant is generally used in a less specific or more generalized structural association therewith.
• Exemplary adjuvants and/or adjuvant combinations may be selected from the group consisting of mineral salts, including aluminum salts, such as aluminum phosphate and aluminum hydroxide (alum) (e.g., AlhydrogelTM, Superfos, Denmark) and calcium phosphate; RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion, whereby any of the 3 components MPL, TDM or CWS may also be used alone or combined 2 by 2; toll-like receptor (TLR) agonists, including, for example, agonists of TLR-1 (e.g.
• tri-acyl lipopeptides tri-acyl lipopeptides
• agonists of TLR-2 e.g. peptidoglycan of gram-positive bacteria like streptococci and staphylococci; lipoteichoic acid]; agonists of TLR-3 (e.g. double-stranded RNA and their analogs such as poly 1:C); agonists of TLR-4 (e.g. lipopolysaccharide (endotoxin) of gram-negative bacteria like Salmonella and E. coli ); agonists of TLR-5 (e.g. flagellin of motile bacteria like Listeria ); agonists of TLR-6 (e.g.
• TLR-2 peptidoglycan and certain lipids diacyllipopeptides
• TLR-7 e.g. single-stranded RNA (ssRNA) genomes of such viruses as influenza, measles, and mumps; and small synthetic guanosine-base antiviral molecules like loxoribine and ssRNA and their analogs
• agonists of TLR-8 e.g. binds ssRNA
• agonists of TLR-9 e.g. unmethylatedCpG of the DNA of the pathogen and their analogs
• agonists of TLR-10 function not defined
• TLR-11- e.g.
• MPL® monophosphoryl lipid A
• D-MPL 3 De-O-acylatedmonophosphoryl lipid A
• OM-174 E.
• coli lipid A derivative OM triacyl lipid A derivative, and other MPL- or lipid A-based formulations and combinations thereof, including MPL®-SE, RC-529 (Dynavax Technologies), AS01 (liposomes+MPL+QS21), AS02 (oil-in-water PL+QS-21), and AS04 (Alum+MPL)(GlaxoSmith Kline, Pa.), CpG-oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motifs, double-stranded RNA, polyinosinic:polycytidylic acid (poly I:C), and other oligonucleotides or polynucleotides optionally encapsulated in liposomes; oil-in-water emulsions, including AS03 (GlaxoSmith Kline, Pa.), MF-59 (microfluidized detergent stabilized squalene oil-in-water emulsion; Novartis
• coli heat-labile enterotoxin LT
• immune-adjuvants including cytokines, such as IL-2, IL-12, GM-CSF, Flt3, accessory molecules, such as B7.1
• mast cell (MC) activators such as mast cell activator compound 48/80 (C48/80); water-insoluble inorganic salts; liposomes, including those made from DNPC/Chol and DC Chol; micelles; squalene; squalane; muramyl dipeptides, such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as found in U.S. Pat. No.
• adjuvant potency may be enhanced by combining multiple adjuvants as described above, including combining various delivery systems with immunopotentiating substances to form multi-component adjuvants with the potential to act synergistically to enhance antigen-specific immune responses in vivo.
• immunopotentiating substances include the above-described adjuvants, including, for example, MPL and synthetic derivatives, MDP and derivatives, oligonucleotides (CpGetc), ds RNAs, alternative pathogen-associated molecular patterns (PAMPs) ( E.
• coli heat labile enterotoxin coli heat labile enterotoxin
• flagellin saponins (QS-21 etc)
• small molecule immune potentiators SMIPs, e.g., resiquimod [R848]
• cytokines e.g., IL-12, IL-12, and chemokines.
• Control release preparations may be achieved through the use of polymers to complex or absorb the oligosaccharides, oligosaccharide conjugates, and/or adjuvants.
• Controlled delivery may be effected by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the method of incorporation in order to control release.
• Another possible method to control the duration of action by controlled release preparations is to incorporate the compounds of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
• a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
• these agents instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate)-microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
• colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nano
• oligosaccharide compositions of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable vehicle (or diluents).
• a pharmaceutically acceptable vehicle or diluents.
• Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences, supra.
• a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of the above-described compounds together with a suitable amount of protein carrier and/or vehicle.
• the immunogenic or immunoprotective compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection.
• An aqueous composition for parenteral administration may include a solution of the immunogenic component(s) dissolved or suspended in a pharmaceutically acceptable vehicle or diluent, preferably a primarily aqueous vehicle.
• Pharmaceutically acceptable vehicles or diluents may include water, saline, including neutral saline solutions buffered with phosphate, Tris, glycerol, ethanol, and the like.
• An aqueous composition may be formulated as a sterile, pyrogen-free buffered saline or phosphate-containing solution, which may include a preservative or may be preservative free. Suitable preservatives include benzyl alcohol, parabens, thimerosal, chlorobutanol, and benzalkonium chloride, for example.
• Aqueous solutions are preferably approximately isotonic, and its tonicity may be adjusted with agents such as sodium tartrate, sodium chloride, propylene glycol, and sodium phosphate.
• auxiliary substances required to approximate physiological conditions including pH adjusting and buffering agents, tonicity adjusting agents, wetting or emulsifying agents, pH buffering substances, and the like, including sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamineoleate, etc. may be included with the vehicles described herein.
• compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered.
• the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
• the preparation of such pharmaceutical compositions is within the ordinary skill in the art, and may be guided by standard reference books such as Remington's Pharmaceutical Science, supra, which is incorporated herein by reference.
• compositions may be formulated in a solid or liquid form for oral delivery.
• nontoxic and/or pharmaceutically acceptable solid protein carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
• a pharmaceutically acceptable nontoxic composition may be formed by incorporating any of the normally employed excipients, including those protein carriers previously listed, and a unit dosage of an active ingredient, that is, one or more compounds of the invention, whether conjugated to a protein carrier or not.
• Topical application of antibodies to an airway surface can be carried out by intranasal administration (e.g., by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally).
• Topical application of the antibodies to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the antibodies as an aerosol suspension, and then causing the subject to inhale the respirable particles.
• respirable particles of a pharmaceutical formulation including both solid particles and liquid particles
• Methods and apparatuses for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed.
• Oral administration may be in the form of an ingestable liquid or solid formulation.
• compositions may be formulated in an aerosol for nasal administration.
• the immunogenic compounds are preferably supplied in finely divided form along with one or more surfactant(s) and/or propellant(s).
• the surfactant will be nontoxic, and preferably soluble in the propellant.
• Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
• Mixed esters such as mixed or natural glycerides may be employed.
• the surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%.
• the balance of the composition is ordinarily propellant.
• a protein carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
• the concentration of the immunogenic oligosaccharides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 0.1% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., and in accordance with the particular mode of administration selected.
• a human unit dose form of the compounds and composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable protein carrier, preferably an aqueous protein carrier, and is administered in a volume of fluid that is known by those of skill in the art to be used for administration of such compositions to humans, and is adjusted according to commonly understood principles for a particular subject to be treated.
• the invention provides a unit dosage of the vaccine components of the invention in a suitable amount of an aqueous solution, such as 0.1-3 ml, preferably 0.2-2 mL.
• the immunogenic and immunoprotective compositions of the present invention may be administered to any animal species at risk for developing an infection by P. aeruginosa.
• the treatment may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of treatment may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months.
• suitable treatment schedules include: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient to elicit the desired responses expected to reduce disease symptoms, or reduce severity of disease.
• the amounts effective for inducing an immune response or providing protective immunity will depend on a variety of factors, including the oligosaccharide composition, conjugation to a protein carrier, inclusion and nature of adjuvant(s), the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician.
• the amounts may generally range for the initial immunization (that is for a prophylactic administration) from about 1.0 ⁇ g to about 5,000 ⁇ g of carbohydrate antigen for a 70 kg patient, (e.g., 1.0 ⁇ g, 2.0 ⁇ g, 2.5 ⁇ g, 3.0 ⁇ g, 3.5 ⁇ g, 4.0 ⁇ g, 4.5 ⁇ g, 5.0 ⁇ g, 7.5 ⁇ g, 10 ⁇ g, 12.5 ⁇ g, 15 ⁇ g, 17.5 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 75 ⁇ g, 100 ⁇ g, 250 ⁇ g, 500 ⁇ g, 750 ⁇ g, 1,000 ⁇ g, 1,500 ⁇ g, 2,000 ⁇ g, 2,500 ⁇ g, 3,000 ⁇ g, 3,500 ⁇ g, 4,000 ⁇ g, 4,500 ⁇ g or 5,000 ⁇ g).
• a primary dose may optionally be followed by boosting dosages of from about 1.0 to about 1,000 of carbohydrate antigen (e.g., 1.0 ⁇ g, 2.0 ⁇ g, 2.5 ⁇ g, 3.0 ⁇ g, 3.5 ⁇ g, 4.0 ⁇ g, 4.5 ⁇ g, 5.0 ⁇ g, 7.5 ⁇ g, 10 ⁇ g, 12.5 ⁇ g, 15 ⁇ g, 17.5 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 75 ⁇ g, 100 ⁇ g, 250 ⁇ g, 500 ⁇ g, 750 ⁇ g, 1,000 ⁇ g, 1,500 ⁇ g, 2,000 ⁇ g, 2,500 ⁇ g, 3,000 ⁇ g, 3,500 ⁇ g, 4,000 ⁇ g, 4,500 ⁇ g or 5,000 ⁇ g) pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific T cell activity in the patient's blood
• the present invention contemplates the use of single- and multi-valent glycoconjugate vaccines comprising any of the synthetic oligosaccharides described herein.
• the identification of a single oligosaccharide antigen eliciting a cross-reactive immune response can facilitate development of a single-antigen vaccine candidate active against all common P. aeruginosa bacterial serotypes and/or strains.
• the present invention further contemplates multi-antigen glycoconjugate vaccines comprising a plurality of any of the synthetic oligosaccharides described herein so as to provide protection against a single serotype or serotype subtype of P. aeruginosa or against a plurality of serotypes or serotype subtypes of P. aeruginosa .
• the invention provides a composition containing two, three, four or more different oligosaccharide antigens according to Formula 1 b.
• the immunogenic compositions comprising a compound of the invention may be suitable for use in adult humans or in children, including young children or others at risk for contracting an infection caused by a LOS-expressing bacterial species.
• a composition may be administered in combination with other pharmaceutically active substances, and frequently it will be administered in combination with other vaccines as part of a childhood vaccination program.
• compositions for administration may beneficially include multiple oligosaccharide- or oligosaccharide conjugates that elicit an immune response to a plurality of different epitopes so as to provide increased protection against a single strain or serotype of P. aeruginosa or against a plurality of strains or serotypes of P. aeruginosa .
• compositions may be administered whereby a prime immunization with one or multiple antigen conjugates is followed by boosting events with one or more cross-reactive core conjugates according to the present invention.
• the invention provides diagnostic antibodies, as well as pharmaceutical compositions comprising one or more anti-LOS antibody(ies) or a functional fragment(s) thereof, and a physiologically acceptable vehicle. Methods for generating these antibodies are further described below.
• compositions may be used in a method for providing passive immunity against P. aeruginosa infections.
• a pharmaceutical antibody composition may be administered to an animal subject, preferably a human, in an amount sufficient to prevent or attenuate the severity, extent of duration of the infection by one or more strains or serotypes of P. aeruginosa.
• the administration of one or more antibodies may be either prophylactic (prior to anticipated exposure to a bacterial infection) or therapeutic (after the initiation of the infection, at or shortly after the onset of the symptoms).
• the dosage of the one or more antibodies will vary depending upon factors as the subject's age, weight and species. In general, the dosage of the antibody may be in a range from about 1-10 mg/kg body weight.
• the antibody is a humanized antibody of the IgG or the IgA class.
• the route of administration of the one or more antibodies may be oral or systemic, for example, subcutaneous, intramuscular or intravenous.
• kits useful for diagnosing, treating, and/or preventing a P. Aeruginosa infection may include one or more containers holding the diagnostic or pharmaceutical compositions of the invention.
• the kits may also include other container(s) containing, for example, one or more solutions necessary or convenient for the particular diagnostic or pharmaceutical use.
• the container means can be made of glass, plastic or foil and can be a vial, bottle, pouch, tube, bag, etc.
• the kit may also contain written information, such as procedures for carrying out the present invention or analytical information, such as the amount of reagent contained in the container(s).
• the present invention provides compositions and methods for inducing production of antibodies for use in assay development, including their use as detection agents and serum screening tools.
• Antisera to LOS-conjugates may be generated in New Zealand white rabbits by 3-4 subcutaneous injections over 13 weeks. A pre-immune bleed may generate about 5 mL of baseline serum from each rabbit. A prime injection (10 ⁇ g antigen equivalent) may be administered as an emulsion in complete Freund's adjuvant (CFA). Subsequent injections (5 ⁇ g antigen equivalent) may be given at three week intervals in incomplete Freund's adjuvant (IFA). Rabbits may be bled every two weeks commencing one week after the third immunization.
• CFA complete Freund's adjuvant
• IFA incomplete Freund's adjuvant
• serum Approximately 25-30 mL of serum per rabbit may be generated from each bleeding event and frozen at ⁇ 80° C. Serum may be analyzed by ELISA against the corresponding LOS-conjugate as described below. In addition, antisera from later bleeds may be affinity purified as further described below.
• the oligosaccharides and antibodies of the present invention can be used as diagnostic reagents for detecting P. aeruginosa LOS antigens or antibodies thereagainst, which are present in biological samples.
• the detection reagents may be used in a variety of immunodiagnostic techniques, known to those of skill in the art, including ELISA- and microarray-related technologies.
• these reagents may be used to evaluate antibody responses, including serum antibody levels, to immunogenic oligosaccharide conjugates.
• the assay methodologies of the invention typically involve the use of labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, and/or secondary immunologic reagents for direct or indirect detection of a complex between an antigen or antibody in a biological sample and a corresponding antibody or antigen bound to a solid support.
• labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules
• secondary immunologic reagents for direct or indirect detection of a complex between an antigen or antibody in a biological sample and a corresponding antibody or antigen bound to a solid support.
• Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
• substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
• a solid support is first reacted with a first binding component (e.g., an antigen or antibody in accordance with the present invention) under suitable binding conditions such that the first binding component is sufficiently immobilized to the support.
• a first binding component e.g., an antigen or antibody in accordance with the present invention
• mobilization to the support can be enhanced by first coupling the antibody or oligosaccharide to a protein with better binding properties, or that provides for immobilization of the antibody or antigen on the support without significant loss of antibody binding activity or specificity.
• Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art.
• BSA bovine serum albumin
• KLH keyhole limpet hemocyanin
• Other molecules that can be used to bind antibodies the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like. Such molecules and methods of coupling these molecules are well known to those of ordinary skill in the art and are described in, e.g., U.S. Pat. No. 7,595,307 and U.S. Pat. Appl. No. US 2009/0155299.
• Disaccharide S (11 g) was suspended in THF: MeOH (200 mL total, 10:1 v:v) and cooled to 0° C. Ammonia was bubbled through the reaction mixture for 15 min. The reaction mixture was capped and allowed to warm to room temperature with stirring over 12 h. The reaction mixture was purged with nitrogen and concentrated in vacuo to a thick syrup. Intermediate T was isolated as a mixture of ⁇ - and ⁇ -isomers and used in the next step without purification
• TMSOTf trimethylsilyl trifluoromethanesulfonate
• Compound HH can be prepared according to the methods of Paulsen, H. et al Liebigs Ann. Chem. 1992, 735 and is a key intermediate that may be used for the synthesis of building block LL.

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